Mifepristone alters the morphology of cancer cells in a dose-related manner
We previously observed, using phase contrast microscopy, that cytostatic concentrations of MF cause elongation of the cytoplasm in various cancer cell types [13]. Now, double fluorescence labeling allows to simultaneously appreciate that both, morphological changes and reduced cellular density, induced by MF, are dose-related (Additional file 1: Figure S1A-D). The double fluorescence staining emphasizes the position of the nucleus within the cell while highlighting the pronounced cytoplasmic stretching along all the cell lines.
Mifepristone attenuates migration and invasion of cancer cells
Cancer-cell migration is critical for distant metastases. The cell’s ability to rearrange its cytoskeleton and propel itself forward is necessary before invasion and movement throughout various tissues can occur. Cells that cannot move towards a source of nutrition (blood vessels, for instance) will likely not survive. Thus, due to the importance of this process, we studied the impact of morphological changes triggered by cytostatic concentrations of MF [13] (Table 1) on cellular migration.
The migration of SKOV-3 cells in a wound healing assay was impaired at both 18 and 30-h time-points. At 30 h, vehicle-treated cells had substantially closed the wound, whereas MF-treated cells were unable to move any farther than they had after 18 h, leaving the wound relatively at the same initial size (Fig. 1c, d).
In MDA-MB-231 cells treated with MF, migration was impaired by 18 h and significantly inhibited by 30 h. Whereas vehicle-treated cells nearly tripled the number of cells within the wounded area, the number of MF-treated cells in the wounded area remained similar to 0 h (Fig. 1e, f). The LNCaP prostate cancer cells were by far the slowest moving cells. However, even when they migrated at a slow pace, MF treatment still significantly attenuated migration at both 18 h and 30 h (Fig. 1g). Finally, the migration of MF-treated U87MG cells was significantly reduced, at both times studied, when compared to vehicle-treated cells (Fig. 1h).
The migration of MF-treated SKOV-3 cells through the Boyden chamber insert diminished significantly when compared to vehicle-treated cells by 18 and 24 h (Fig. 2a). The migration of MDA-MB-231 cells (Fig. 2b) and LNCaP cells (Fig. 2c) was significantly inhibited by MF pre-treatment at all evaluated time-points. U87MG cells were the most aggressive, with the largest number of cells migrating through the insert by 9 h, compared to the other cell lines. However, as early as 9 h, and at both 18 and 30-h time-points, the inhibitory effect of MF treatment was significant in comparison to vehicle-treated cells (Fig. 2d). In summary, the inhibitory effect of MF on cell migration was observed in all cell lines, regardless of their basal migratory capacity.
SKOV-3, LNCaP, and U87MG cells were similar in their invasive capacities, and MF significantly diminished the invasion of all cells after both 18 and 24 h (Fig. 2e, g, h). The breast cancer cell line, MDA-MB-231, was the most invasive, yet it also showed significant inhibition of invasion by MF at both 18 and 24 h (Fig. 2f).
Validation of the wound healing assay in cancer cells upon MF treatment using double fluorescence staining
The inhibitory effect of MF on the migration of each cancer cell line in a wound healing assay was validated with the addition of Alexa Fluor®-594 Phalloidin and DAPI, to stain the actin cytoskeleton and nucleus, respectively. All four cell lines were studied using the same variation of the migration method.
The migration of SKOV-3 cells was significantly reduced at 24 h. In this case, MF-treated cells barely migrated after 24 h, compared to 0 h (Fig. 3a, b). These results are consistent with the results previously described, in which MF-treated cells at 30 h were unable to move any farther than they had after 18 h (Fig. 1c). U87MG and MDA-MB-231 were the most migratory cell lines, almost completely closing the wound after 24 h; although cells treated with MF were capable of migrating, their migration rate was significantly diminished when compared to cells treated with vehicle in both cell lines (Fig. 3c, d). LNCaP was by far the slowest migrating cell line, barely closing the wound after 24 h. The average wound width of MF-treated cells was relatively the same at 0 and 24 h; MF-induced reduction of wound healing was yet found to be significant compared with untreated cells at this time point (Fig. 3e).
Enhancement of the Boyden chamber assays in cancer cells upon MF treatment using double fluorescence staining
Upon enhancement of the staining of migratory cells using Alexa Fluor®-594 Phalloidin and SYTOX® Green, the effect of MF remained consistent in all cell lines. SKOV-3 cells were found to be the most migratory and demonstrated a large increase in the number of migrated cells between 6 and 24 h. MF was able to significantly inhibit the migration of these cells as early as 6 h after the start of the experiment (Fig. 4a, b). U87MG and MDA-MB-231 cells were once again found to be highly migratory through this assay, although these experiments showed MDA-MD-231 cells to be more migratory than U87MG cells. In both cases, cells were found to be quite migratory at 6 h, however MDA-MB-231 demonstrated a much larger increase in the number of migratory cells at 24 h compared to U87MG. In both cell lines, MF was able to attenuate the migration at 6 and 24 h, however this effect was most drastically observed in MDA-MB-231, where cells were unable to migrate more than they had after 6 h, even after 24 h (Fig. 4c, d). Finally, LNCaP was once again the slowest migrating cell line. In this case, similar to MDA-MB-231, at 6 h, MF-treated cells were unable to migrate any farther than they had at 24 h; however, this inhibition was only significant when compared to vehicle-treated cells at 24 h (Fig. 4e).
To study and validate the invasion and the effect of MF on each cell line, the Boyden chamber assay was repeated but with the addition of a layer of ECM in the upper chamber. Once again, the effect of MF was consistent. SKOV-3 and U87MG had a similar invasive rate, and in both cell lines, the inhibitory effect of MF was observed after 6 and 24 h (Fig. 4f-h). MDA-MB-231 and LNCaP cells both had a slower invasive capacity. However, MF was still able to significantly inhibit their invasiveness (Fig. 4i-j).
The addition of double fluorescence staining to migration assays unveils varying migration patterns in cancer cells
A crucial event of cancer metastasis is the migration of cancer cells from the primary tumor to secondary, distant sites. Two distinct patterns of migration have been described: single-cell and collective-cell migration (rev. in [17,18,19,20]). In single-cell migration, cells migrate individually and invade surrounding tissues independent of each other. Collective-cell migration involves groups of cells adherent to one another, migrating and invading surrounding tissues as multicellular aggregates. By enhancing the wound healing and Boyden chamber assays with the addition of a double fluorescence staining—labeling actin cytoskeleton and nuclear DNA—it is possible to better observe such migration patterns.
When observing SKOV-3 cells in a wound healing assay, it was noticed that cells treated with vehicle formed a sheet as they closed the wound, suggesting collective-cell migration (Fig. 5a, i; and Fig. 3b). SKOV-3 cells having migrated through the pores in the Boyden chamber seemed to recreate this phenomenon, as cells were observed to be covering the entire surface of the image, almost always touching one another (Fig. 5a, iii). When SKOV-3 cells were treated with MF, the cells in the front leading edge were more isolated and less adherent to one another. This change in organization can be visualized through both wound healing and Boyden Chamber methods (Fig. 5a, ii and iv; and Fig. 3b). In both wound healing and Boyden chamber migratory assays, U87MG and MDA-MB-231 were observed to express a similar migration pattern, with vehicle-treated cells loosely organized and with very little attachment to one another (Fig. 5b, i and iii; and c, i and iii). This behavior indicates that these cancer cell lines most likely undergo single-cell migration. When both U87MG and MDA-MB-231 cells were treated with MF, although the pattern remained similar to that of vehicle-treated cells, there was an obvious decrease in the number of migratory cells (Fig. 5b, ii and iv; and ,c ii and iv). Finally, LNCaP cells demonstrated a mixed migration pattern (Fig. 5d, i and iii). In the wound healing assay, some LNCaP leading cells show a mix of collective and individual migration (Fig. 5d,i). The collective migration was more obvious when the cells traversed the pores of the polycarbonate membrane of the Boyden chamber and appear as tri-dimensional clusters (Fig. 5d, iii). When LNCaP cells were treated with MF, in the wound healing assay, cells were observed to be less cohesive; yet, the front leading edge retained grouped cells (Fig. 5d, ii). Such grouping or clustering was also maintained when cells migrated through the pores of the Boyden chamber (Fig. 5d, iv).